The broad goals of the proposed program are to study mechanisms involved in axonal pathfinding during development and regeneration. Of specific interest is the signaling that translates a chemorepellent's action on the growth cone into collapse. Our data indicate that generation of eicosanoids by 12/15-lipoxygenase is necessary and sufficient (at least partially) for semaphorin 3A-induced collapse. Other results suggest that synthesis of these eicosanoids is part of a cascade involving, upstream, cytosolic phospholipases A2 (cPLA/2) and, downstream, protein kinase C (PKC) and adhesion site proteins, whose phosphorylation may trigger adhesion site disassembly and detachment. In a series of cell biological studies we will test the hypothesis that this pathway regulates detachment of the growth cone periphery during collapse. Experiments will be focused on the effects of semaphorin 3A. In cultures of responsive neurons we will study growth structure and adhesion. In isolated growth cone preparations enriched in the semaphorin 3A receptor, neurophilin-1, we will analyze cPLA2 activation, eicosanoid synthesis and PKC stimulation, as well as phosphorylation of adhesion site proteins. These studies are expected (i) to provide new insights into the little understood signalling mechanisms activated by repellants and (ii) to establish a novel signalling pathway that is believed to trigger disassembly of growth cone adhesion sites. The analysis of the mechanisms of growth cone pathfinding and repulsion is fundamental to our understanding of nervous system development, and insight into the action of chemorepellants on growth cones is likely to reveal new options for promoting nerve regeneration in the adult CNS.